Project Summary: Many genetic diseases that affect the central nervous system (CNS) remain untreatable due to a lack effective small molecule drugs or biologics. Targeting the genetic underpinnings of these diseases with somatic cell gene editing would therefore be particularly impactful, but its successful implementation will require methods to safely and efficiently deliver genes and gene editing machinery throughout the CNS. AAVs are the state-of-the-art vehicles for in vivo gene transfer because they can provide safe and long lasting in vivo gene expression. AAVs are the only gene therapy vectors that have been approved for direct administration to humans by regulatory agencies in both the US and Europe. Moreover, in 2017, AAVs became the first vehicle used as part of an early phase clinical trial to evaluate the safety of in vivo gene editing. Despite their impressive preclinical and clinical safety record, naturally occurring AAVs tested to date lack the efficiency required for gene delivery across most organ systems, including the CNS. To address the need for better vehicles for CNS gene delivery, we recently used directed evolution and a new cell type-specific in vivo selection method to engineer several novel AAVs, most notably AAV-PHP.B and AAV-PHP.eB, that have, for the first time, made it possible to noninvasively transfer genes to the majority of neurons and astrocytes throughout the adult mouse CNS. Here, we aim to build upon the success of this selection approach by engineering AAVs that enable efficient gene transfer throughout the CNS of multiple species, including nonhuman primates. The AAVs we develop will be evaluated in several species for their ability to provide CNS-wide transgene expression and targeted genome editing in neurons, and improved AAV variants will be shared with the scientific community. Successful completion of this project, which involves pairing the new AAVs with next-generation gene editing technologies, will provide support for evaluating the safety of CNS gene editing in human trials.